Genomic analysis of increased host immune and cell death

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author ManuscriptTo study the virulence and pathogenesis of the 1918 influenza virus, mice were infectedintranasally with a contemporary human influenza A/Texas/36/91 H1N1 virus (Tx91), Tx91virus containing the 1918 HA and NA genes (2:6 1918), the 1918 HA, NA, M, NP and NSgenes (5:3 1918), or the reconstructed 1918 (H1N1) recombinant virus (r1918). Mice infectedwith r1918 showed the most severe weight loss and the earliest mean time of death of all virusestested (Supplementary Information). Moreover, titration of virus recovered from lung tissueshowed that mice infected with r1918 had about tenfold higher levels of virus on days 1 and 3after infection than those infected with 2:6 1918 or 5:3 1918. By day 5 after infection therewas a slight decrease in the lung titres of r1918-infected mice, and mice infected with 2:6 1918,5:3 1918 and r1918 showed similar lung titres of about 107 50% embryo-infective doses perml. Our results were consistent with those in previous reports3: most mice infected with r1918were dead by day 5 after infection.Previous studies showed increased lung pathology in mice infected with influenza virusesbearing genes from the 1918 virus3–5. A more detailed histological analysis of mice infectedwith 5:3 1918, 2:6 1918 or r1918 viruses revealed differences in pathology during the courseof infection. Whereas mice infected with Tx91 reassortant virus had minimal to mild diffusehistiocytic alveolitis without neutrophils or lesions in the bronchi or bronchioles (Fig. 1a), miceinfected with 5:3 1918, 2:6 1918 or r1918 viruses had pulmonary lesions that consisted of mildto severe necrotizing bronchitis with accompanying moderate to severe alveolitis andassociated moderate to severe alveolar oedema (Fig. 1b–f). However, the character of thealveolitis varied between the 1918 viruses and the sampling periods. At day 3, the r1918-infected mice had acute moderate to severe diffuse alveolitis with a prominent neutrophiliccomponent and severe alveolar oedema (Fig. 1b). For some lungs, alveolitis was multifocalwith intense neutrophilic infiltration that distended the alveoli (Fig. 1c). By contrast, miceinfected with 5:3 1918 and 2:6 1918 viruses had moderate to severe alveolitis, but histiocytespredominated over neutrophils and the alveolar oedema was only moderately severe (Fig. 1d,e). At day 5 the pulmonary response in r1918-infected mice had changed to subacute and was

more severe with diffuse alveolitis retaining the severe oedema, but changing to a

predominance of histiocytes with fewer neutrophils (Fig. 1f). The bronchial and bronchiolar

lesions were similar to those at day 3. However, in some r1918-infected lungs, an acute

inflammatory response continued, as was evident from multiple scattered foci of intense

neutrophilic alveolitis. In contrast, some mice infected with 5:3 1918 and 2:6 1918 viruses

showed slightly less alveolitis, alveolar oedema, bronchitis and peribronchitis on day 5 than

on day 3. These results show that the pathology induced by r1918 was distinct from that

observed in mice infected with 5:3 1918 and 2:6 1918 viruses, indicating that cooperation

between the individual 1918 viral proteins might be required for the induction of maximum

pathology.

To understand why 1918 influenza virus showed extreme virulence in mice, we used gene

expression analysis to provide a global view of the host response in lungs of infected mice.

Expression microarray analysis was performed by taking equal masses of total RNA isolated

from lungs of mice infected with strain Tx91, 2:5 1918, 5:3 1918 or r1918 and comparing them

with a common reference sample prepared from lungs of mock-infected mice. Lungs from

three mice were harvested for each virus on days 1, 3 and 5 after infection (total n = 36).

Initially, our analysis was directed to genes associated with the activation of key immune cells

known to be important for the response to influenza virus infection, including T helper 1

(TH1), natural killer, macrophage and neutrophil cells (shown in Fig. 2). This analysis revealed

a significant increase in the expression of genes associated with these immune cell populations

in lungs of mice infected with r1918. Expression patterns of cytokines, chemokines and

apoptosis-related genes showed the most significant and earliest activation during r1918

infection (Supplementary Information). The increased expression of immune-cell-related